This project will refine factors that define the protocol for mating compatible strains of Aspergillus fumigatus in order to decrease the time required to complete the meiotic cycle. A. fumigatus is an important mould pathogen of immunocompromised hosts. The frequency of infections is increasing and the morbidity and mortality attributable to invasive disease remains unacceptably high. Although molecular genetic techniques have enabled researchers to make good progress in investigating factors involved in the biology of the organism and the pathogenesis of disease, the addition of classical genetics to the armamentarium of the research community would markedly enhance our ability to study this organism. The primary barrier to the widespread use of classical genetics is the prolonged period required for completion of the mating cycle. The six month incubation renders this approach unfeasible for routine use. The hypothesis is that by methodically evaluating environmental conditions under which mating takes place, the current system can be optimized. In Specific aim 1, the environmental conditions, including medium, water, temperature, atmosphere, and light, will be optimized to reduce the time required for mating between isolates of A. fumigatus. Our goal is to achieve a 4 - 6 week window for the completion of the cycle, and our preliminary data shows that the required time has already been reduced by 1/3 - 1/2. In the second specific aim, commonly used markers for the molecular manipulation of A. fumigatus will be evaluated for their effect upon mating. Drug markers, auxotrophic markers, and other factors, such as ablation of NHEJ, will be evaluated. Completion of these studies should provide the A. fumigatus research community with a powerful new tool to use in research on this important pathogen.